CN109705203B - Protein related to plant type and coding gene and application thereof - Google Patents
Protein related to plant type and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a protein related to plant types and a coding gene and application thereof. The protein related to the plant type disclosed by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is the protein of sequence 1; A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function; A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). Experiments prove that the protein related to the plant type and the gene thereof can regulate the plant type, particularly the plant height of the plant, the expression of the gene can be inhibited to obviously reduce the plant height of the plant, the protein related to the plant type and the gene thereof can be used for cultivating the plant with reduced plant height, and have important application value for effectively regulating and controlling the plant type of the plant through genetic breeding and genetic engineering methods.
Description
Technical Field
The invention relates to a protein related to plant types, a coding gene and application thereof in the field of biotechnology.
Background
Rice, as an important food crop, lives over one third of the world's population. With the growing population, the reduction of the cultivated land area and the deterioration of the environment, rice production is under greater and greater pressure in terms of ensuring yield. The plant type of rice is an important agronomic trait related to yield, and mainly depends on several aspects of plant height, leaf type, tillering number, tillering angle, spike morphology and the like. Therefore, the discovery and the utilization of the related gene for controlling the rice plant type have important significance for rice breeding and production.
Cell division and expansion are the most fundamental physiological processes in plant growth and development, and plant cells complete cell proliferation by continuously forming new cell plates and cell walls, and are accompanied by a plurality of complex physiological processes in the process of division and expansion, wherein vesicle transport of an intracellular endomembrane system is responsible for the transport of a plurality of synthetic raw materials.
Endocytosis of plant cells affects not only the most basic cell differentiation function, but also plant growth and development, transmission of hormonal signals, and communication with the external environment, such as nutrient transport, tolerance to toxic substances, resistance to pathogenic bacteria, and the like. The clathrin-mediated endocytosis is the most main route of plant cell endocytosis, and the clathrin initially forms vesicles on cell membranes, recognizes and wraps specific endocytic goods on the cell membranes, and then transports substances into cells. Compared with extensive and intensive research on animal cells, the research on the endocytosis mechanism of plant cells at present is mainly based on the research model of animal cells and the hypothesis of related theories, and the plant cells still have great research space due to the existence of the division mechanism which is different from that of the animal cells.
Disclosure of Invention
The invention aims to solve the technical problem of how to regulate and control the plant type of a plant, particularly the plant height.
In order to solve the technical problems, the invention firstly provides any one of the following applications of the plant type related protein derived from rice or a substance for regulating the activity or the content of the plant type related protein;
D1) regulating and controlling plant types of plants;
D2) preparing a plant type regulating product;
D3) cultivating short-stalk plants;
D4) preparing and cultivating short-stalk plant products;
the plant type related protein (the name of which is DGSP1) is A1), A2) or A3 as follows:
A1) the amino acid sequence is the protein of sequence 1;
A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;
A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).
In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing may be labeled as shown in the following table.
Table: sequence of tags
Label (R) | Residue of | Sequence of |
Poly-Arg | 5-6 (typically 5) | RRRRR |
Poly-His | 2-10 (generally 6) | HHHHHH |
FLAG | 8 | DYKDDDDK |
Strep-tag II | 8 | WSHPQFEK |
c-myc | 10 | EQKLISEEDL |
The DGSP1 protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 1 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
The DGSP1 protein in A2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.
The gene encoding DGSP1 protein in A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID NO.2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end a coding sequence of the tag shown in the above table. Wherein, the DNA molecule shown in the sequence 2 encodes DGSP1 protein shown in the sequence 1.
The invention also provides any one of the following uses of the biological material related to DGSP 1;
D1) regulating and controlling plant types of plants;
D2) preparing a plant type regulating product;
D3) cultivating short-stalk plants;
D4) preparing and cultivating short-stalk plant products;
the biomaterial is any one of the following B1) to B40):
B1) a nucleic acid molecule encoding DGSP 1;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector comprising the nucleic acid molecule of B1);
B4) a recombinant vector comprising the expression cassette of B2);
B5) a recombinant microorganism comprising the nucleic acid molecule of B1);
B6) a recombinant microorganism comprising the expression cassette of B2);
B7) a recombinant microorganism containing the recombinant vector of B3);
B8) a recombinant microorganism containing the recombinant vector of B4);
B9) a transgenic plant cell containing the nucleic acid molecule of B1);
B10) a transgenic plant cell containing the expression cassette of B2);
B11) a transgenic plant cell containing the recombinant vector of B3);
B12) a transgenic plant cell containing the recombinant vector of B4);
B13) transgenic plant tissue comprising the nucleic acid molecule of B1);
B14) transgenic plant tissue comprising the expression cassette of B2);
B15) transgenic plant tissue containing the recombinant vector of B3);
B16) transgenic plant tissue containing the recombinant vector of B4);
B17) a transgenic plant organ containing the nucleic acid molecule of B1);
B18) a transgenic plant organ containing the expression cassette of B2);
B19) a transgenic plant organ containing the recombinant vector of B3);
B20) a transgenic plant organ containing the recombinant vector of B4);
B21) a nucleic acid molecule that reduces the expression level of DGSP 1;
B22) an expression cassette comprising the nucleic acid molecule of B21);
B23) a recombinant vector comprising the nucleic acid molecule of B21);
B24) a recombinant vector comprising the expression cassette of B22);
B25) a recombinant microorganism comprising the nucleic acid molecule of B21);
B26) a recombinant microorganism comprising the expression cassette of B22);
B27) a recombinant microorganism containing the recombinant vector of B23);
B28) a recombinant microorganism containing the recombinant vector of B24);
B29) a transgenic plant cell containing the nucleic acid molecule of B21);
B30) a transgenic plant cell containing the expression cassette of B22);
B31) a transgenic plant cell containing the recombinant vector of B23);
B32) a transgenic plant cell containing the recombinant vector of B24);
B33) transgenic plant tissue comprising the nucleic acid molecule of B21);
B34) transgenic plant tissue comprising the expression cassette of B22);
B35) transgenic plant tissue containing the recombinant vector of B23);
B36) transgenic plant tissue containing the recombinant vector of B24);
B37) a transgenic plant organ containing the nucleic acid molecule of B21);
B38) a transgenic plant organ containing the expression cassette of B22);
B39) a transgenic plant organ containing the recombinant vector of B23);
B40) a transgenic plant organ containing the recombinant vector of B24).
In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) or B15):
b11) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;
b12) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;
b13) a DNA molecule shown in a sequence 3 in a sequence table;
b14) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) or b13) and encoding DGSP 1;
b15) a cDNA or DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) or b14) and encodes DGSP 1;
B21) the nucleic acid molecule may be a DNA fragment represented by formula I below:
SEQ forward-X-SEQ reverse (I);
the SEQ forward direction is a partial fragment of sequence 2 or the full length thereof;
the sequence of the SEQ reverse direction is complementary to the sequence of the SEQ forward direction in a reverse direction;
and the X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to the SEQ forward direction and the SEQ reverse direction in sequence.
In the above application, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
The SEQ forward direction may specifically be the nucleotide sequence at position 400-799 of SEQ ID NO 2.
The nucleotide sequence of the DNA fragment shown in the formula I is a sequence 4 in a sequence table, the nucleotide sequence from the 1 st to the 400 th site in the sequence 4 is a nucleotide sequence from the 400 th-through 799 th site in the sequence 2, the nucleotide sequence from the 429 th-through 1616 th site is a nucleotide sequence of Arabidopsis FAD2 intron, and the nucleotide sequence from the 1623 th-through 2045 th site is a nucleotide sequence which is reversely complementary to the nucleotide sequence from the 400 th-through 799 th site in the sequence 2.
The nucleotide sequence encoding DGSP1 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of DGSP1 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode DGSP1 protein and have the function of DGSP1 protein.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown as coding sequence 1 shown as sequence 2 or 3 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO4Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO4Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO4Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO4Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO4Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.
The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.
In the above applications, the expression cassette containing a nucleic acid molecule encoding DGSP1 protein (DGSP1 gene expression cassette) described in B2) refers to a DNA capable of expressing DGSP1 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of DGSP1 gene, but also a terminator that terminates transcription of DGSP1 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter T7lac, a constitutive promoter of cauliflower mosaic virus CaMV35S, a tomato ribulose-1, 5-bisphosphate carboxylase minor (rbcs) gene promoter; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: t7 terminator, Agrobacterium tumefaciens nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (1985), Nature, 313: 810; Rosenberg et al (1987), Gene,56: 125; Guerineau et al (1991), mol.Gen.Genet,262: 141; Propudfoot (1991), Cell,64: 671; Sanfacon et al, GenesDev., 5: 141; Mogen et al (1990), plantatcell, 2: 1261; Munroe et al (1990), Gene, 91: 151; Ballad et al (1989), nucleic acids, Res.17: 7891; Joshi et al (1987), Resclei, 9615: 9627).
The recombinant vector containing the DGSP1 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pET-28a, pCAMBIA2301, pSP72, pROKII, pBin438, pCAMBIA1302, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co., Ltd.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.
In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.
The existing RNA interference vector can be used for constructing a recombinant vector containing a DNA molecule for reducing the expression level of DGSP1, such as pLHRNAi.
B23) The recombinant vector can be pLHRNAi-DGSP 1. The pLHRNAi-DGSP1 is a recombinant vector obtained by replacing a DNA sequence between SacI and SnaBI recognition sites (recognition sequences) of pLHRNAi with a DNA fragment shown in a sequence 4.
In the above application, the microorganism may be yeast, bacteria, algae or fungi. The bacteria can be derived from Escherichia (Escherichia), Erwinia (Erwinia), Agrobacterium (Agrobacterium) such as Agrobacterium tumefaciens EHA105, Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc.
In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.
In the above application, the plant type may be plant height.
In the above application, the plant may be M1) or M2) or M3) or M4):
m1) monocotyledonous or dicotyledonous plants;
m2) gramineous plants;
m3) plants of the genus oryza;
m4) rice.
The invention also provides any one of the following methods:
x1) to obtain a target plant with reduced plant height compared with the acceptor plant, wherein the method comprises the steps of reducing the content of DGSP1 in the acceptor plant, or reducing the activity of DGSP1 in the acceptor plant, or inhibiting the expression of a gene coding for DGSP1 in the acceptor plant;
x2), which comprises reducing the content of DGSP1 in a receptor plant, or reducing the activity of DGSP1 in the receptor plant, or inhibiting the expression of a coding gene of DGSP1 in the receptor plant, so as to obtain a target plant with reduced plant height compared with the receptor plant, and realize the reduction of the plant height.
The recipient plant contains the gene encoding DGSP 1.
In the above method, inhibiting the expression of a gene encoding DGSP1 in a recipient plant can be achieved by introducing B21) said nucleic acid molecule into said recipient plant.
B21) Said nucleic acid molecule can be obtained by introducing a recombinant vector containing the nucleic acid molecule according to B21) into said recipient plant.
B21) The nucleic acid molecule can be realized by introducing the recombinant vector of B23) or B24) into the recipient plant.
In the method, the encoding gene of DGSP1 can be modified as follows and then introduced into a receptor plant to achieve better expression effect:
1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the amino acid sequence of the gene encoding DGSP1 of the present invention may be changed to conform to plant preferences while maintaining the amino acid sequence, depending on the preferred codons of the recipient plant; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;
2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;
3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;
4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;
5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.
The gene encoding DGSP1 can be introduced into a recipient plant by using a recombinant expression vector containing the gene encoding DGSP 1.
Both the recombinant vector and the recombinant vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition)).
The plant of interest is understood to comprise not only the first generation plants in which the DGSP1 protein or the gene encoding it has been altered, but also the progeny thereof. For the plant of interest, the gene may be propagated in the species, or transferred into other varieties of the same species, including commercial varieties in particular, using conventional breeding techniques. The plant of interest includes seeds, callus, whole plants and cells.
In the above method, the recipient plant may be M1) or M2) or M3) or M4):
m1) monocotyledonous or dicotyledonous plants;
m2) gramineous plants;
m3) plants of the genus oryza;
m4) rice.
The invention also provides the following products of Y1) or Y2):
y1) B21) the nucleic acid molecule;
y2) a product for regulating plant type, said product comprising DGSP1 or said biological material.
The product for regulating plant type of the plant can take DGSP1 or the biological material as an active ingredient, and can also combine DGSP1 or the biological material with substances with the same functions as the active ingredient.
In the product, the plant type can be the plant height.
In the above product, the plant may be M1) or M2) or M3) or M4):
m1) monocotyledonous or dicotyledonous plants;
m2) gramineous plants;
m3) plants of the genus oryza;
m4) rice.
Experiments prove that the plant type related protein and the gene thereof can regulate and control the plant type, particularly the plant height of the plant. Inhibiting the expression of the plant type related protein gene can obviously reduce the plant height of the plant, and proves that the plant type related protein and the gene thereof play an important role in regulating and controlling the plant height of the plant. The invention not only provides a basis for further clarifying the molecular mechanism of plant types of plants, but also provides new gene resources and breeding resources for plant breeding. The plant type related protein, the gene thereof and the nucleic acid molecule for inhibiting the gene expression can be used for cultivating plants with reduced plant height; the transgenic plant with the reduced DGSP1 gene expression, which is obtained by the invention, can be used as a new plant seed material for researching the molecular mechanism of plant dwarfing and finding more genes for regulating and controlling the plant height development of plants. The invention has important application value for effectively regulating and controlling the plant type of the plant by utilizing the gene resource through genetic breeding and genetic engineering methods.
Drawings
FIG. 1 shows the transcript level detection of DGSP1 gene in wild type rice and transgenic interfering families.
FIG. 2 is a phenotypic observation of RNA interference transgenic rice with reduced expression level of DGSP1 gene.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
The rice Kitaake (also called wild-type rice, abbreviated WT) in the following examples is described in GaoH, ZhengXM, WanJM., et al, Ehd4encodes a Novel and Oryza-gene-specific regulator of photo-technological flowability in rice PLOS GENET.2013,9(2): e1003281) which is publicly available from the institute of crop science of the Chinese academy of agricultural sciences, and is used only for repeating the experiments related to the present invention and is not applicable for other uses.
The expression vector pLHRNAi used in the following examples is pLHRNAi in Chinese patent 201110055864.X, and the public can obtain the biological material from the research institute of crop science of Chinese academy of agricultural sciences, and the biological material is only used for repeating the related experiments of the invention and cannot be used for other purposes.
The Agrobacterium used in the examples described below was Agrobacterium tumefaciens EHA105(Agrobacterium tumefaciens EHA105) (New Agrobacterium hel plasmids for gene transfer to plants. hood, ElizabethE; Gelvin, Stanton B; Melchers, LeoS; Hoekema, Andre. transfer research,2(4): p.208-218(1993)), which was publicly available from the research of crop science of the Chinese academy of agricultural sciences, and which was used only for the repetition of the experiments related to the present invention and was not used for other purposes.
Example 1 plant type-related protein DGSP1 can regulate and control the plant height of rice
The embodiment provides a plant type related protein derived from rice Kitaake (Oryza sativa var. Kitaake), which is named as DGSP1, wherein the amino acid sequence of DGSP1 in the rice Kitaake is sequence 1 in a sequence table, the coding sequence (namely CDS sequence) of DGSP1 gene is sequence 2 in the sequence table, and the genome sequence of DGSP1 gene is sequence 3 in the sequence table.
Construction of RNA interference vector for reducing DGSP1 gene expression level
1. Obtaining of DGSP1 gene expression interference fragment
(1) Total RNA of 14-day seedlings of rice Kitaake (Oryzasativa) was extracted using an RNAprep plant total RNA extraction kit (Tiangen Biochemical technology, Beijing) Co., Ltd.) and reverse-transcribed to obtain cDNA.
(2) And (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and a primer pair consisting of DGSP1-sense-F and DGSP1-sense-R to obtain a fragment 1(SEQ forward direction).
DGSP1-sense-F:5'-CTAGGTACCAGGCCTGAGCTCGCGTTAGGTAGGAACATTG-3';
DGSP1-sense-R:5'-GACGTAGGGGCGATAGAGCTCGAGCTTCTAATGATACCCC-3'。
(3) And (2) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and a primer consisting of DGSP1-antisense-F and DGSP1-antisense-R to obtain a fragment 2(SEQ reverse direction).
DGSP1-antisense-F:5'-TCTTAGAATTCCCGGGGATCCGCGTTAGGTAGGAACATTG-3';
DGSP1-antisense-R:5'-CGTTACGTAGTCGACGGATCCGAGCTTCTAATGATACCCC-3'。
2. Construction of DGSP1 Gene RNA interference vector (recombinant expression vector pLHRNAi-DGSP1)
(1) The expression vector pLHRNAi is digested by restriction endonuclease SacI to obtain a linear expression vector pLHRNAi, and the linear fragment is recovered. Integrating the fragment 1 obtained in the step 1 (2) to a linear expression vector pLHRNAi (the concrete method refers to a clone infusion kit (please ensure the white) instruction) by adopting a homologous recombination directional cloning method to obtain a homologous recombination product 1, then transferring the homologous recombination product 1 to an escherichia coli DH5 alpha competent cell, culturing overnight at 37 ℃, and marking the obtained recombination vector with a correct sequence as pLHRNAi-sense-DGSP 1.
(2) The recombinant vector pLHRNAi-sense-DGSP1 is cut by restriction endonuclease SnaBI enzyme to obtain a linear vector pLHRNAi-sense-DGSP1, and the linear vector is recovered. Integrating the fragment 2 obtained in the step 1 (3) onto a linear vector pLHRNAi-sense-DGSP1 by adopting a homologous recombination directional cloning method (the concrete method refers to the clone infusion kit instruction), obtaining a homologous recombination product 2, transferring the homologous recombination product 2 into an escherichia coli DH5 alpha competent cell, culturing overnight at 37 ℃, and marking the obtained recombinant vector with a correct sequence as pLHRNAi-DGSP 1.
(3) Sequencing is carried out on the recombinant vector pLHRNAi-DGSP1, and the result shows that the recombinant vector pLHRNAi-DGSP1 is formed by inserting a double-stranded DNA fragment shown by a nucleotide sequence from 400 th to 799 th bit from 5 'end of SEQ ID No.2 into the SacI enzyme cutting site of the expression vector pLHRNAi in the forward direction, inserting a double-stranded DNA fragment which is reversely complementary to the double-stranded DNA fragment shown by the nucleotide sequence from 400 th to 799 th bit from 5' end of SEQ ID No.2 into the SnaBI enzyme cutting site, thus successfully replacing the DNA sequence between the SacI and the SnaBI recognition sites (recognition sequences) of the pLHRNAi with a DNA fragment shown by a sequence 4 and a reverse complementary DNA fragment thereof, and connecting the two DNA fragments by partial DNA fragments in the vector.
Constructing RNAi interference transgenic plant with reduced DGSP1 gene expression level and identifying transgenic plant
One) construction of transgenic plants
The rice Kitaake is mediated and transformed by a recombinant vector pLHRNAi-DGSP1 through Agrobacterium tumefaciens EHA105, and an empty vector control plant is constructed by utilizing the expression vector pLHRNAi according to the same method, and the specific method is as follows:
1. and (3) introducing the recombinant vector pLHRNAi-DGSP1 obtained in the step one into the Agrobacterium tumefaciens EHA105 by a heat shock method to obtain the recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector pLHRNAi-DGSP 1. The recombinant Agrobacterium tumefaciens EHA105 was cultured at 28 ℃ for 16 hours, and the cells were collected. The bacterial cells were diluted with N6 liquid medium (Sigma, catalog No. C1416) containing 100. mu.M acetosyringone to obtain diluted bacterial solution, OD600 of which was about 0.5.
2. Mixing mature embryogenic callus of rice Kitaake cultured for one month with the diluted bacterial solution obtained in step 1, infecting for 30min, drying the surface bacterial solution of callus by filter paper, transferring into N6 solid co-culture medium (N6 mixed medium formula is potassium nitrate (2800mg/L), ammonium sulfate (463mg/L), potassium dihydrogen phosphate (400mg/L), magnesium sulfate (MgSO 2)4·7H2O) (185mg/L), calcium chloride (CaCl)2·2H2O) (165mg/L), disodium ethylene diamine tetraacetate (37.3mg/L), ferrous sulfate (FeSO)4·7H2O) (27.8mg/L), manganese sulfate (MnSO)4·H2O) (4.4mg/L), zinc sulfate (ZnSO)4·7H2O) (1.5mg/L), boric acid (1.6mg/L), potassium iodide (0.8mg/L), vitamin B1 (thiamine hydrochloride) (1.0mg/L), vitamin B6 (pyridoxine hydrochloride) (0.5mg/L), nicotinic acid (0.5mg/L), glycine (2.0mg/L), sucrose (20000 mg/L). Weighing 24.1g of N6 mixed culture medium, heating and stirring to dissolve in 1000ml of distilled water, adjusting pH to 5.8 with sodium hydroxide, and autoclaving at 115 ℃ for 20 minutes to obtain N6 solidAnd (5) culturing the culture medium. ) Co-culturing at 24 deg.C for 3d to obtain co-cultured callus.
3. The callus after the co-culture treatment in step 2 was inoculated on N6 solid selection medium (a medium obtained by adding hygromycin to N6 solid selection medium, the mass concentration of hygromycin in the N6 solid selection medium being 150mg/L) containing hygromycin at a mass concentration of 150mg/L for the first selection.
4. And (3) picking the healthy callus on the 16 th day from the first screening, transferring the healthy callus to an N6 solid screening culture medium (a culture medium obtained by adding hygromycin to an N6 solid culture medium, wherein the mass concentration of the hygromycin in the N6 solid screening culture medium is 200mg/L) containing the hygromycin, culturing, and carrying out secondary screening, wherein the secondary screening is carried out once every 15 days and is carried out for 1 time in total, and the obtained healthy callus is the resistant callus.
5. And (3) selecting the resistant callus obtained in the step (4), transferring the resistant callus to a differentiation medium (the differentiation medium: 6-BA 2mg, NAA 0.2mg and N6 mixed medium is 4g, hydrolyzed casein is 1g, inositol is 0.1g, cane sugar is 25g, sorbitol is 2.4g, agar powder is 7g, deionized water is supplemented to 1L) containing hygromycin with the mass concentration of 150mg/L, carrying out differentiation culture, culturing for 45d at 24 ℃ (the height of the overground part of the plant is about 15cm), opening a bottle mouth, hardening seedlings for 3 days, and then transplanting to a greenhouse for culture, namely a transferred pLHRNAi-DGSP1 plant (T0 generation).
II), RNAi interference transgenic plant PCR identification with reduced DGSP1 gene expression level
Extracting genome DNA of T0 generation seedlings of the transferred pLHRNAi-DGSP1 plant and seedlings of the rice Kitaake plant (abbreviated as WT) obtained in the step one), and performing PCR molecular detection by using primers 1390-F (5'-TGCCTTCATACGCTATTTATTTGC-3') and FAD2-R (5'-GAAGCGACGGACCTGGAGAT-3') to identify positive seedlings, wherein the obtained plants of 562bp PCR products are positive transgenic plants, and the rice Kitaake plant (wild type) cannot obtain 562bp PCR products. Two positive transgenic plants are taken and named as a pLHRNAi-DGSP1 transferred plant RNAi-1 (RNAi-1 plant for short) and a pLHRNAi-DGSP1 transferred plant RNAi-2 (RNAi-2 plant for short) respectively.
Third, RNAi interference transgenic plant with reduced DGSP1 gene expression level identification of DGSP1 gene expression level
Respectively extracting RNA of the RNAi-1 plant (interference family 1), the RNAi-2 plant (interference family 2) and the rice Kitaake plant (wild type) leaf obtained in the second step, setting internal reference as ubiptin, and carrying out fluorescence quantitative PCR by using internal reference primers UBI-F and UBI-R and DGSP1 gene specific quantitative primers DGSP1-qRT-F and DGSP1-qRT-R to detect the change of the expression level of the DGSP1 gene of each plant. The results (fig. 1) show that the expression levels of the DGSP1 gene in RNAi-1 and RNAi-2 plants were significantly reduced compared to the expression level of the DGSP1 gene in rice Kitaake (wild type), and that there was no significant difference in the expression levels of the DGSP1 gene in the empty vector control plant and rice Kitaake. The primers are as follows:
UBI-F:5’-GCTCCGTGGCGGTATCAT-3’;
UBI-R:5’-CGGCAGTTGACAGCCCTAG-3’;
DGSP1-qRT-F:5’-GATTGGTCGCAATGCAGTAGCC-3’;
DGSP1-qRT-R:5’-GATTGGTCGCAATGCAGTAGCC-3’。
RNAi interference transgenic plant phenotype identification with reduced DGSP1 gene expression level
And (3) respectively planting the RNAi-1 plant (interference family 1), the RNAi-2 plant (interference family 2), the empty carrier control plant and the rice Kitaake plant (wild type) obtained in the step two in a sequential experimental base of the institute of crop science of the Chinese academy of agricultural sciences, and observing the phenotypic difference of each plant in the whole growth period. The observation results are shown in fig. 2, compared with the rice Kitaake (wild type) plant, the RNAi-1 (interfering family 1) plant and the RNAi-2 (interfering family 2) plant both have the phenotype of plant height dwarfing, the average plant heights of the rice Kitaake (wild type), the RNAi-1 (interfering family 1) plant and the RNAi-2 (interfering family 2) plant are 69.61 +/-3.53 cm, 43.33 +/-2.16 cm and 38.42 +/-1.73 cm respectively in the rice mature period, and the heights of the empty vector control plant and the rice Kitaake plant are not obviously different. Thereby proving that the DGSP1 gene participates in controlling the growth and development of rice plant types.
<110> institute of crop science of Chinese academy of agricultural sciences
<120> plant type-related protein, and coding gene and application thereof
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 570
<212> PRT
<213> Rice (Oryza sativa)
<400> 1
Met Asp Arg Leu Arg Ala Gly Ser Pro Val Tyr Gly Arg Gln Arg Ser
1 5 10 15
Gly Ser Ser Thr Gly Ser Ser Ser Pro Gly Gly Val Ser Pro Ser His
20 25 30
His Arg Ser Ser Ser Thr Ser Ser Ala Ala Ser Ala Ala Ala Gly Leu
35 40 45
Gly Gly Gly Val Ser Asn Val Arg Arg Thr Gln Asn Val Ala Ala Arg
50 55 60
Ala Ala Ala Ala Arg Leu Ala Gln Val Met Ala Ser Gln Ser Ala Ala
65 70 75 80
Ala Ala Ala Gly Arg Asp Asp Asp Asp Asp Asp Asp Asp Tyr Ala Asn
85 90 95
Asp His Pro Pro Ala Pro Pro Pro Ala Arg Phe Gly Ser Ala Arg Pro
100 105 110
Ala Ala Ala His Gly Ser Asn Gly Val Ser Leu Leu Gly Arg Thr Ala
115 120 125
Arg Ser Pro Ser Pro Ala Leu Gly Arg Asn Ile Val Glu Pro Pro Pro
130 135 140
Thr Val Arg Ser Thr Ser Ala Gly Arg Pro Ala Val Ala Ser Arg Pro
145 150 155 160
Thr Thr Thr Val Val Pro Pro Ile Lys Thr Ser Thr Thr Leu Arg Thr
165 170 175
Pro Ser Pro Ile Pro Pro Val Ala Val Glu Pro Pro Val Asp Arg Ser
180 185 190
Arg Gln Lys Arg Phe Asp Thr Gly His Leu Asn Ser Arg Glu Ser Thr
195 200 205
Pro Lys Arg Glu Ala Ser Ala Leu Gln Asp Glu Leu Asp Ile Leu Gln
210 215 220
Glu Glu Asn Glu Ser Val Leu Glu Lys Leu Arg Leu Ala Glu Glu Arg
225 230 235 240
Cys Glu Glu Ala Glu Ala Arg Ala Lys Glu Leu Glu Lys Gln Val Ala
245 250 255
Ala Leu Gly Glu Gly Val Ser Leu Glu Ala Arg Leu Leu Ser Arg Lys
260 265 270
Glu Ala Ala Leu Lys Gln Arg Glu Ala Ala Leu Lys Ala Ala Arg Glu
275 280 285
Ser Lys Asp Gly Lys Asp Gly Glu Val Thr Thr Leu Lys His Glu Leu
290 295 300
Asp Cys Ala Lys Glu Glu Val Val Thr Ala Met Glu Gln Leu Lys Glu
305 310 315 320
Ala Glu Thr Glu Thr Lys Ala Leu Arg Ser Met Thr Gln Arg Met Ile
325 330 335
Leu Thr Gln Glu Glu Met Glu Glu Val Val Leu Lys Arg Cys Trp Leu
340 345 350
Ser Arg Tyr Trp Gly Leu Ala Val Gln Tyr Gly Val Tyr Pro Glu Ile
355 360 365
Ala Val Ser Lys His Glu His Trp Ser Ser Leu Ala Pro Leu Pro Leu
370 375 380
Glu Val Val Leu Ser Ala Gly Gln Lys Ala Lys Glu Glu Pro Leu Lys
385 390 395 400
Gln Gly Glu Asp Asp Ala Gln Arg Arg Asn Lys Leu Val Arg Asp Met
405 410 415
Ser Asp Val Met Gly Glu Gly Asn Ile Glu Ser Met Leu Ser Val Glu
420 425 430
Met Gly Leu Arg Glu Leu Ser Ser Leu Lys Val Glu Asp Ala Val Val
435 440 445
Val Ala Leu Gly Gln His Arg Arg Pro Ser Ile Val Arg Gln Phe Thr
450 455 460
Ser Asp Phe Lys Ser Pro Gly Glu Pro Lys Phe Leu Glu Ala Phe Asp
465 470 475 480
Leu Ser His Glu Glu Ala Glu Asp Val Ser Phe Lys Gln Ala Trp Leu
485 490 495
Ile Tyr Phe Trp Arg Arg Ala Lys Thr His Gly Ile Glu Glu Asp Ile
500 505 510
Ala Glu Glu Arg Leu Gln Phe Trp Ile Gly Arg Asn Ala Val Ala Pro
515 520 525
Thr Ser His Asp Ala Ile Asp Val Glu Arg Gly Leu Thr Glu Leu Arg
530 535 540
Lys Leu Gly Ile Glu Gln Gln Leu Trp Glu Gly Ser Arg Ala Asp Ile
545 550 555 560
Asp Glu Asp Ser Ser Ala Ile Glu Asn His
565 570
<210> 2
<211> 1713
<212> DNA
<213> Rice (Oryza sativa)
<400> 2
atggaccgcc tccgcgcggg gagccccgtc tacgggcggc agcggagcgg cagcagcacg 60
ggctcctcct ccccgggcgg cgtctccccg tcccaccacc gctcctcctc cacctcctcc 120
gccgcctccg ccgccgcggg gctgggcggc ggcgtctcca acgtgcgccg cacgcagaac 180
gtcgcggcgc gggcggccgc cgcgaggctg gcccaggtca tggcgtcaca gagcgccgcg 240
gccgccgcgg gccgcgacga cgacgacgac gacgacgact acgccaatga ccacccgccc 300
gcccctcccc ccgcgaggtt cggctccgcg cgccccgccg cggcgcacgg cagcaacggc 360
gtctcgttgc tcggccgcac cgcgagatct ccctcccctg cgttaggtag gaacattgta 420
gagccacctc ctactgtccg ctcaacatca gctgggaggc ctgccgttgc ttcacggccg 480
accaccacgg tggttccacc gatcaaaacc agcacgacat tgcggacacc atcgcctatt 540
ccacctgtgg ctgtggagcc tccagtggac cgcagtcggc agaaaaggtt tgatacaggg 600
catctcaact ccagagaatc aacaccaaaa agggaggcgt ctgcacttca agatgagctt 660
gatatactac aagaggagaa tgagagtgtt ctagaaaagc tacgacttgc tgaagaaaga 720
tgtgaagaag cagaagctag agccaaggag cttgagaaac aggtagctgc ccttggggaa 780
ggggtatcat tagaagctcg cctcttgagc aggaaagaag ctgcccttaa acagagggag 840
gctgcactga aggctgcaag ggaatcaaaa gatggtaaag atggggaagt gacaacacta 900
aagcatgaac tggattgtgc caaagaagag gttgtaacag ctatggagca gctaaaggaa 960
gcagagactg aaacaaaggc cctccggtcc atgactcaga gaatgatctt aacccaagag 1020
gaaatggaag aggtggtcct taaaaggtgc tggctttcac gttattgggg cttagcggtt 1080
caatacggag tttatcccga gattgcggta tcaaagcatg aacattggtc atcattagct 1140
cctcttcccc ttgaggttgt tctctctgct ggtcaaaagg ctaaggagga acctctcaaa 1200
caaggtgagg atgacgccca aaggagaaat aaacttgtgc gagatatgag tgatgtaatg 1260
ggagaaggca atatagagag catgctctca gttgaaatgg ggcttagaga gctttcatca 1320
ttgaaggtgg aagatgctgt tgtggttgca cttggtcaac accggagacc tagtatagtt 1380
cggcaattca catcagattt taaatcacct ggtgaaccta aattcttgga ggcgtttgat 1440
cttagccatg aagaggcaga agatgttagc tttaagcagg catggcttat atacttctgg 1500
agaagagcta aaactcatgg cattgaggaa gacattgctg aagaacggct tcagttctgg 1560
attggtcgca atgcagtagc cccaacttct catgatgcta tagatgtgga gcgaggtcta 1620
acagagctca ggaaactggg catagagcag caactgtggg aaggatcacg tgcagatatc 1680
gatgaagatt catcggcaat tgagaatcac tag 1713
<210> 3
<211> 7123
<212> DNA
<213> Rice (Oryza sativa)
<400> 3
acctccttcc cacttctcaa cctcgcctcg ccatctccgc gccgcggccc agatccctcc 60
ggcgagcgcc gccgccgcca ccgcctccgc catggaccgc ctccgcgcgg ggagccccgt 120
ctacgggcgg cagcggagcg gcagcagcac gggctcctcc tccccgggcg gcgtctcccc 180
gtcccaccac cgctcctcct ccacctcctc cgccgcctcc gccgccgcgg ggctgggcgg 240
cggcgtctcc aacgtgcgcc gcacgcagaa cgtcgcggcg cgggcggccg ccgcgaggct 300
ggcccaggtc atggcgtcac agagcgccgc ggccgccgcg ggccgcgacg acgacgacga 360
cgacgacgac tacgccaatg accacccgcc cgcccctccc cccgcgaggt tcggctccgc 420
gcgccccgcc gcggcgcacg gcagcaacgg cgtctcgttg ctcggccgca ccgcgagatc 480
tccctcccct gcggtaaatt ctgcttcccc cggatctctc gctgatctcg ctacccgctg 540
cgtctcaact taggatttcg atggtttgaa ctgtcgcggt gcacgaatga gtcatagttg 600
gtcaatcgga agtcaaattg gttctgaagc atgcttgagc agatgaattt gctagtgaag 660
ctagtttaca tctcgccaat gtcagtccaa ttctatagcg atgccttctg aattgtaaaa 720
attgggagaa gtgatgaatg cattgggact tctggctgct tcgtgatttg atcaccgctt 780
atcctgtgcc cttctgctaa tttgatctcc tcatgttttc attagttagg taggaacatt 840
gtagagccac ctcctactgt ccgctcaaca tcagctggga ggcctgccgt tgcttcacgg 900
ccgaccacca cggtggttcc accgatcaaa accagcacga cattgcggac accatcgcct 960
attccacctg tggctgtgga gcctccagtg gaccgcagtc ggcagaaaag gtacgtcaat 1020
gacattcccc agatctgttg catggtctgc agcattctct gtgttttcta tacttaaaat 1080
agcattcctt aagttctact tctcttagga tatattaaaa tgaatgcatg gttaattgta 1140
ctagcaaccc caccaaatcg agcagaaatg agttattttt actgcttgcg ctgtttttca 1200
gaagaagtga tgttggtagt tcacaacatt aactaaactg ctcttgataa agggatgtat 1260
ttggttggtg gcgttgttag taaaaaaaga tgcattgata tcataaggtt gaaccaacag 1320
ccctatgcca aactatgtgt tcagttttgg tcggtatagt ggagagccct cttatcttgg 1380
gcgtaatgga ttgaaaagtt ttgagttcaa taatacatgc ttattaagga tgggtagtaa 1440
attattatgc gtccgaaatc ttaatcgctt acctaaattg tgaaatctga tatttgattg 1500
aatcaattgg tcagcatctt ggggttgtgg gtgtgcctct tggccagttt cctattaccg 1560
cgctttgctg aactgcgccg cagaatctat caaattatac ataattttta cctattctag 1620
caaatgattt cttttattgt gctgaaaagg gcattgctga ctagttatta ataataacaa 1680
aaaattcggc cccaagggga aagatctccc ctgaggtatt tcaattaaga agaagcacct 1740
gaaaccgagg ccccagaaag gccacaaaag ctggtcctcc tacatgggga gccgccccct 1800
atggatcggg ccttaatcca tgctttgagg ctcttgcccc ctacacaagg acgatagatc 1860
aattcctaac cttgttttgg tagaaccagc gagtgacctt ttttggtgca agaccaagat 1920
ttgaaccctg gtcgttagct tcccactgga aggtatttac catcatgcta caagcacgtt 1980
tacttgctga ctagttaata tatctaggat tagtaaaata aagtgccctt tagtcgtcca 2040
ccatgctaaa cgcacccttt tctcttttaa gtatgttact aggggcacca cattgcttgt 2100
tgcattaaag ttttggaata atcacattat gaccatctgg ccacttctta tagcttgaat 2160
ttctttttcc cattttttca aatgttccta aaaataggca tcaggtaagt tatttacttt 2220
ttttttttgc ctcaggtttg atacagggca tctcaactcc agagaatcaa caccaaaaag 2280
ggaggcgtct gcacttcaag atgaggtttt ccttattctc tataaactag ttatacgtat 2340
aatatatttt agctcatggt tctgtcacga actttgacct agaacttttt gaggagtgtt 2400
tatcagatgt ttttaagact aatgcaatgc tgtcctacag cttgatatac tacaagagga 2460
gaatgagagt gttctagaaa aggtgaaata gcaaccatag agctgtttta taggcaatct 2520
agtacttgat tttatactga tctttaacaa attattttct gaatcttgat tgacagctac 2580
gacttgctga agaaagatgt gaagaagcag aagctagagc caaggagctt gagaaacagg 2640
tgagataatc ttatattaaa cattgtttta tctttttttt tgtattatga taaattggac 2700
atgtgcacta gacattgagt ctatctctag ttctcactac agtttttctt ttggcgggaa 2760
tttttcacta caatttaaac ttgaatttga attattggaa cgaatattct cctttacttg 2820
aggaggtgtt cattacactg agcattttgt atctttggtt agctaaggtt atatttgttg 2880
atatgcaggt agctgccctt ggggaagggg tatcattaga agctcgcctc ttgagcaggt 2940
ttgcatcatg tttggatatg catgtttcac aaattcagaa ctgatcatag actcataacc 3000
taacattgtc atgattattg gcaggaaaga agctgccctt aaacagaggg aggttagttt 3060
ttcttcttgt gttttaccta tggatagctg gcatgccaat cttgtagtac tgtttttttt 3120
atcttagaat ttatatgtaa aatttggaag tgttagaatt ttagtgtttt tcttgttgtg 3180
aaaacaaaag gatgaaacat tttggaattt catttgatct tttttctgct ttatttgtct 3240
tgaaatgcac atgcggtact atagtcatgc taggttctta ttgtaaaaaa aaaatctgaa 3300
cctgaaggat atgctggagt acataccttc caattttacc agaaaaataa agaggatatt 3360
tcttacccat caacgaacac tttatataat gctttttgtc tacaggctgc actgaaggct 3420
gcaagggaat caaaagatgg taaagatggg gaagtgacaa cactaaagca tgaactggat 3480
gtaaatatac ttcctaccaa tcaaaatatg gaagtagtgt tggtttgtat atggtgtttt 3540
atttgaccat gttttcttct catgctttga atagtgtgcc aaagaagagg ttgtaacagc 3600
tatggagcag ctaaaggaag cagagactga aacaaaggcc ctccggtcca tgactcagag 3660
aatgatctta acccaagagg aaatggttag tctgatttca gtactcccat tttttaattg 3720
cttgatatag attttgatct atttgcaacc ttatgctagg aagaggtggt ccttaaaagg 3780
tgctggcttt cacgttattg gggcttagcg gttcaatacg gtaagttgct aagagtattt 3840
ctgtaagaaa tgcttttagt gttatcttgt ataagctgtt ctgtaatcat gctctctaga 3900
caaaataaaa tcaaagctta tcatgtcaaa aggatgttag ttatcatggt gaaacaaaag 3960
aaataaatta tgttgcacat ttgttatttg ttgttctctg ttgaaaggat gttagttatc 4020
atgtgattac tttgtttggt taaatatcaa tagtgtgagc tagttgctgg aaattggaaa 4080
ggcaaacatt ttttttgtga cttatatgca atttggttta ttgcgtttac agaaatggtt 4140
ttcaacatag tttttgtttc gcagtttatc tttagatgct gataattcac gttcacacta 4200
gacctgagaa tttggtctgg tctgaatgca agtttaaggc tggtttggtc tcagaaaatg 4260
aggccaatgg ccaaacagtc catactccat cctgactatg tatatggcta ggtgtcactt 4320
agattagggc ctagggggcc tgaaagtaac tagttgtgac cctggtctgg tctcgaaaat 4380
gcttaattct agtttgcaca tgatgttgaa atgtactgtt aactttttcc ctgtccgtga 4440
tttatctgat ggcccattaa taacttgaag gagtttatcc cgagattgcg gtatcaaagc 4500
atgaacattg gtcatcatta gctcctcttc cccttgaggt tgttctctct gctggtcaaa 4560
aggctaagga ggaacctctc aaacaaggca tgtctgcatc tttataatat acaagttgta 4620
gatcgtagtt tgttccatgt actccagtta attattcttg gatcttttca agcaggtgag 4680
gatgacgccc aaaggagaaa taaacttgtg cgagatatga gtgatgtaat gggagaaggc 4740
aatatagaga gcatgctctc agttgaaatg gggcttagag agctttcatc attgaaggta 4800
ttccactgcc cttttattgg cagtggttta ttaactttct gttctataga ttctcagaga 4860
tgaaattcca gacacagtac ttgtactcat tctaatgcag agacgaacat ttacagcaaa 4920
aatggcacaa cagtaattct tctaatatcc tctagagttt ttttttatgg agtgttctct 4980
gaaatacgga agtgtgaaca gtatttgcca ggctttacct tacttccact gtaatagagc 5040
actaagtaat cattcttttt gtactgagta tatttgcatg tttggacaag tgacaaacca 5100
tatgtgcttt ataaatacag gttttttttt ctaggcttcc ttatcacttc tctcctcact 5160
agttattgtt ctgtaggtgg aagatgctgt tgtggttgca cttggtcaac accggagacc 5220
tagtatagtt cggcaattca catcaggttt gcacttcttt tatcatttca tatcattctt 5280
gtttaactta catgcgtaat atgatttctt cattggtgct gccttcactc ataaaaaaaa 5340
actaagaaag gtataaattc ttctcttatg cagattttaa atcacctggt gaacctaaat 5400
tcttggaggc gtttggtaat ggttcttccc tttgtctaat agatttatgc aaaacttcca 5460
ctaacagctt gagcttgtgt tgtatgtaaa catttcatcc tgtttctatg agtttaaatt 5520
aaatttgaac tttttgacct attgcagatc ttagccatga agaggcagaa gatgttagct 5580
ttaagcaggt aaacctttca gcgatgtgaa ctatcaaatt catccattta ttacgtgtga 5640
tttatggaat atatattgaa tgcaggcatg gcttatatac ttctggagaa gagctaaaac 5700
tcatggcatt gaggaagaca ttgctgaaga acggcttcag ttctggattg gtcgcaatgc 5760
agtagcccca acttctcatg atgctataga tggtaataag caactttcct tgttgaaaaa 5820
ataacctagc attgggagtt atccacctgt tagttaatga tttatcgtac tatttaacca 5880
tccaacgtcc ctgggatgtc ttttatttgt gcagctcttt tttgacagca tgccccaaca 5940
gttgatcatg tgatgctttt gtagggatca aaccaacttt ctatttcctc cttttattat 6000
ttcttcactg ttgtatttta tctctttgcc ctttactgtc ctctttccca tctgtaatcc 6060
aaagtacggt atagtggtac gaacattatg gtcccgttcg tttctccaac aagagttgga 6120
tgaagattta gattttcgtg gcacactttt gaaaccgcta aacggtgcgt ttcgtgcgaa 6180
aactttctat atggaagttg ttttaaaata tcagattaat ccatttttca agtttgtaat 6240
aattaaaact catttaatca cacgttatta ccacatcgtt ttgcgtgaaa cacttaatct 6300
ttatcttcat cttcatcttc aggagattca aacaccacct atatgtgaga tacttacatg 6360
aaccactatt aatgtgattg atgtgttttt ttttgtctaa acagttatgg gatattgggg 6420
gtatacagaa ggcattttga gtatgccctc atatatatta catgttgcgg tgtgcctata 6480
atctgttttg tttttccaat tgtttttacc tagatttaca cacctcatcc attttttctc 6540
ctatccaaga atgccatcat tttcagtttt ttgctttact gagcactgtt agttttgtta 6600
tctgcagtgg agcgaggtct aacagagctc aggaaactgg gcatagagca gcaactgtgg 6660
gaaggatcac gtgcagatat cgatgaagat tcatcggcaa ttgagaatca ctaggcacat 6720
ctcaaggttg tttatggtac atgattattc caattttgga gaatggaaat gctttttcgc 6780
ggctggtctt gggccattgc catcattgga tccctgaatg gtgctgcaat cttcgggtgt 6840
tgagcaatgg gcagtcttcg ggtgttgagc gatattatgg gcttgtccag agatattgtg 6900
tagtgcatgt gcgagccatt tatcttttgt cgaaatattt ttgtgaaatg tgtaattgtt 6960
ttgtcctggc aatatgatgg tgatctcact tcatccaatt tgtgaatttg tgattacacc 7020
acatgtgtac tgcctgttat tcagtaaaat atacagagag gaagctatgc ttcctgcgac 7080
aaatgagcaa atcataagtg atgattcttt ctggtagtct gcc 7123
<210> 4
<211> 400
<212> DNA
<213> Rice (Oryza sativa)
<400> 4
gcgttaggta ggaacattgt agagccacct cctactgtcc gctcaacatc agctgggagg 60
cctgccgttg cttcacggcc gaccaccacg gtggttccac cgatcaaaac cagcacgaca 120
ttgcggacac catcgcctat tccacctgtg gctgtggagc ctccagtgga ccgcagtcgg 180
cagaaaaggt ttgatacagg gcatctcaac tccagagaat caacaccaaa aagggaggcg 240
tctgcacttc aagatgagct tgatatacta caagaggaga atgagagtgt tctagaaaag 300
ctacgacttg ctgaagaaag atgtgaagaa gcagaagcta gagccaagga gcttgagaaa 360
caggtagctg cccttgggga aggggtatca ttagaagctc 400
Claims (6)
1. Any one of the following applications of the substance for reducing the expression level of the plant type related protein;
D1) cultivating short-stalk rice;
D2) preparing and cultivating short-stalk rice products;
the plant type related protein is A1) or A2) as follows:
A1) the amino acid sequence is the protein of sequence 1;
A2) a1) at the N-terminus or/and the C-terminus.
2. Use of any one of the following biomaterials associated with a plant type-related protein according to claim 1;
D1) cultivating short-stalk rice;
D2) preparing and cultivating short-stalk rice products;
the biomaterial is any one of the following B1) to B20):
B1) a nucleic acid molecule for reducing the expression level of a plant type-related protein according to claim 1;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector comprising the nucleic acid molecule of B1);
B4) a recombinant vector comprising the expression cassette of B2);
B5) a recombinant microorganism comprising the nucleic acid molecule of B1);
B6) a recombinant microorganism comprising the expression cassette of B2);
B7) a recombinant microorganism containing the recombinant vector of B3);
B8) a recombinant microorganism containing the recombinant vector of B4);
B9) a transgenic plant cell containing the nucleic acid molecule of B1);
B10) a transgenic plant cell containing the expression cassette of B2);
B11) a transgenic plant cell containing the recombinant vector of B3);
B12) a transgenic plant cell containing the recombinant vector of B4);
B13) transgenic plant tissue comprising the nucleic acid molecule of B1);
B14) transgenic plant tissue comprising the expression cassette of B2);
B15) transgenic plant tissue containing the recombinant vector of B3);
B16) transgenic plant tissue containing the recombinant vector of B4);
B17) a transgenic plant organ containing the nucleic acid molecule of B1);
B18) a transgenic plant organ containing the expression cassette of B2);
B19) a transgenic plant organ containing the recombinant vector of B3);
B20) a transgenic plant organ containing the recombinant vector of B4).
3. Use according to claim 2, characterized in that:
B1) the nucleic acid molecule is a DNA fragment shown as the following formula I:
SEQ forward-X-SEQ reverse (I);
the SEQ forward direction is a partial fragment of sequence 2 or the full length thereof;
the sequence of the SEQ reverse direction is complementary to the sequence of the SEQ forward direction in a reverse direction;
and the X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to the SEQ forward direction and the SEQ reverse direction in sequence.
4. Use according to claim 3, characterized in that: the SEQ forward direction is the nucleotide sequence at position 400-799 of the sequence 2.
5. Any one of the following methods:
x1), reducing the expression level of the plant type-related protein in the receptor rice, or inhibiting the expression of the plant type-related protein coding gene in the receptor rice, so as to obtain the target rice with reduced plant height compared with the receptor rice;
x2), comprising the steps of reducing the expression level of the plant type related protein in the receptor rice as claimed in claim 1, or inhibiting the expression of the coding gene of the plant type related protein in the receptor rice as claimed in claim 1, so as to obtain the target rice with reduced plant height compared with the receptor rice, and realize the reduction of the plant height of the rice.
6. The method of claim 5, wherein: inhibiting the expression of a gene encoding the rice plant type-associated protein according to claim 1 in recipient rice by introducing the nucleic acid molecule according to B1) of claim 2 or 3 into the recipient rice.
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CN112239492B (en) * | 2019-07-17 | 2022-11-01 | 中国农业科学院生物技术研究所 | Discovery and application of key component SLB1 for controlling size of plant organ |
CN111187342B (en) * | 2019-08-16 | 2022-02-11 | 中国农业科学院作物科学研究所 | ZmG2 application in improving plant strong light stress resistance and yield |
CN111205358B (en) * | 2020-03-04 | 2021-03-23 | 中国农业科学院生物技术研究所 | Os494 protein and coding gene and application thereof |
CN112980873B (en) * | 2021-03-12 | 2022-05-03 | 中国农业科学院作物科学研究所 | Protein related to plant type and coding gene and application thereof |
CN116199753A (en) * | 2021-11-30 | 2023-06-02 | 北京农业生物技术研究中心 | SiDTH2 protein and application of coding gene thereof in improving crop biomass |
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CN108948170A (en) * | 2018-08-20 | 2018-12-07 | 中国农业科学院作物科学研究所 | A kind of plant plant type growth and development GAP-associated protein GAP and its encoding gene and application |
CN109096380A (en) * | 2018-08-24 | 2018-12-28 | 中国农业科学院作物科学研究所 | Application of the OsBICs gene in regulation plant plant height, flowering time |
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CN109112143A (en) * | 2018-08-12 | 2019-01-01 | 华中农业大学 | Control pleiotropic gene SP3 and the application of Plant Height of Rice and fringe type |
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NCBI Reference Sequence: XM_ 015766701.2,PREDICTED: Oryza sativa Japonica Group coiled-coil domain-containing protein SCD2 (LOC4326960), mRNA;None;《GenBank》;20180807;CDS、ORIGIN * |
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